RNA splicing, the biochemical process leading to the removal of introns from precursor RNA, is an essential step in the expression of genetic information. Unlike auto-catalyzed self-splicing reactions, splicing of nuclear pre-mRNA requires ATP and occurs in a complex, dynamic structure called the spliceosome composed of small nuclear ribonucleoproteins. A spliceosome containing unspliced pre-mRNA has been isolated from the yeast prp2 mutant. This functional spliceosome can be isolated and activated to splice upon the addition of ATP and extrinsic splicing factors (ESFs). One of the ESFs is an RNA-dependent ATPase encoded by the yeast PRP2 gene, which binds to the spliceosome and triggers splicing in the presence of ATP. Therefore, PRP2 may either be a key component of the catalytic center or function in a proofreading step. The long-term objectives of this research project are to understand the mechanism of nuclear pre-mRNA splicing through biochemical and genetic analysis of the spliceosomal components and the ESFs. Specifically, the components which constitute the catalytic center in the spliceosome will be investigated. Current studies focus on the identification of the spliceosomal components bound by PRP2 and the RNA activating the ATPase activity of PRP2. Radioactive PRP2 protein will be synthesized in vitro or isolated from yeast cells; splicing extracts will be prepared from prp2 mutants with or without radioactive labeling. The interacting proteins or RNAs can be identified by using UV cross-linking, immunological, and gel electrophoresis techniques. A genetic approach has also been taken to isolate an extragenic suppressor, SRP2; it suppresses the temperature-sensitive phenotype of prp2 and has a cold- sensitive phenotype by itself. The SRP2 gene will be isolated; its role in splicing and its relation with PRP2 will be investigated by molecular genetic techniques. The involvement of specific, important sequences of snRNAs in the catalytic steps of splicing will be analyzed by cleaving these sequences prior to the isolation of the functional spliceosome. The characterization of the heat stable ESF-bn and the search for prp2 proofreading mutants will be given a lower priority. Studies of the important constituents of the spliceosome and the ESFs in yeast may reveal the catalytic center of pre-mRNA splicing and lead to a better understanding of alternative and regulated splicing in higher organisms.